Abstract

BACKGROUND:

Dendritic cells (DCs) are important mediators of anti-tumor immune responses. We hypothesized that an in-depth analysis of dendritic cells and their spatial relationships to each other as well as to other immune cells within tumor draining lymph nodes (TDLNs) could provide a better understanding of immune function and dysregulation in cancer.

METHODS:

We analyzed immune cells within TDLNs from 59 breast cancer patients with at least 5 years of clinical follow-up using immunohistochemical staining with a novel quantitative image analysis system. We developed algorithms to analyze spatial distribution patterns of immune cells in cancer versus healthy intra-mammary lymph nodes (HLNs) to derive information about possible mechanisms underlying immune-dysregulation in breast cancer. We used the non-parametric Mann-Whitney test for inter-group comparisons, Wilcoxon Matched-Pairs Signed Ranks test for intra-group comparisons and log-rank (Mantel-Cox) test for Kaplan Maier analyses.

RESULTS:

Degree of clustering of DCs (in terms of spatial proximity of the cells to each other) was reduced in TDLNs compared to HLNs. While there were more numerous DC clusters in TDLNs compared to HLNs,DC clusters within TDLNs tended to have fewer member DCs and also consisted of fewer cells displaying the DC maturity marker CD83. The average number of T cells within a standardized radius of a clustered DC was increased compared to that of an unclustered DC, suggesting that DC clustering was associated with T cell interaction. Furthermore, the number of T cells within the radius of a clustered DC was reduced in tumor-positive TDLNs compared to HLNs. Importantly, clinical outcome analysis revealed that DC clustering in tumor-positive TDLNs correlated with the duration of disease-free survival in breast cancer patients.

CONCLUSIONS:

These findings are the first to describe the spatial organization of DCs within TDLNs and their association with survival outcome. In addition, we characterized specific changes in number, size, maturity, and T cell co-localization of such clusters. Strategies to enhance DC function in-vivo, including maturation and clustering, may provide additional tools for developing more efficacious DC cancer vaccines.

DCs in TDLNs are organized into smaller, less mature clusters compared to HLNs. (A) Number of DC clusters normalized to the total number of DCs in a node (relative number of DC clusters) (B) average size of a DC cluster in a node (calculated by taking an average of the number of DCs that make up individual DC clusters in a node) and (C) percentage of CD83 positive DCs of all clustered DCs in a node (% mature DCs out of all clustered DCs) were calculated in sections HLN (n=7), NSLN- (n=50) and NSLN+ (n=22). *denotes p<0.05, ** denotes p<0.01 and *** denotes p<0.001.

DC clustering and maturation in NSLN+ nodes correlate with duration of disease-free survival. Using the median value of (A) percentage mature DCs or (B) percentage clustered DCs per node as cutoff, patients having NSLN+ nodes (n=19) were divided into two groups (using exclusion criteria mentioned in the Methods section) with those above the median defined by the solid line and those below the median by the dotted line. Kaplan-Meier curves were plotted showing the difference in duration of disease free survival between the two groups. (C) (i) Linear regression analysis between %clustered DCs and %mature DCs in NSLN+ nodes (n=19) (ii) Percentage mature DCs were calculated among clustered as well as unclustered DCs in NSLN+ nodes (n=19) ns denotes p>0.05.